With the development of synthetic biology and systems biology, overproduction of biomolecules can be achieved by manipulation of regulatory proteins from various sources. FK506 (tacrolimus) is a 23-membered macrolide antibiotic and an important immunosuppressant. Overproduction of FK506 has been achieved by the manipulation of regulatory genes that are located inside the biosynthetic gene cluster, however, little is known on the effect of other regulators on FK506 production. In this study, the effects of three Streptomyces antibiotic regulatory proteins (SARPs) on FK506 production were investigated. These SARPs include bulZ and bulY that are cloned from a FK506 producer Streptomyces sp. KCCM11116P, and one novel SARP family regulatory protein Sx5140 that was obtained from a marine streptomycete S. xinghaiensis. The production titer of the engineered strains exhibited a higher production level compared to that in the control strain (227.99 mg L-1), and overexpression of bulZ resulted in the highest FK506 titer (365.59 mg L-1), which is 1.6-fold of that of the control. Real-time PCR analysis showed that overexpression of bulZ and bulY resulted in increased transcription of tsuR1 which is the gamma-butyrolactone receptor. Variation of transcription levels of fkbN, the positive regulator of FK506 biosynthesis, as well as fkbG, fkbH, fkbI, tcsA, tcsB, tcsD and fkbQ genes that are involved in the FK506 biosynthesis were observed in the SARP overexpression strains compared to those in the control strain. Our results demonstrate the promising potential to utilize alternative regulatory proteins including both endogenous and exogenous ones to enhance the production of useful compounds in microbial strains.